For many years various insulins have been used in the treatment of insulin-dependent Diabetes. It would be natural to treat human beings with human insulin, which is not possible, however, in view of the existing demand. Therefore, for practical reasons bovine and porcine insulin is used. However, to a larger or smaller extent, these insulins give rise to the formation of antibodies in the human body, which i.a. involves a reduced effect of the further insulin treatment.
This disadvantage is supposed to be caused partly by "impurities" in the bovine/porcine insulin partly by the alien nature. The latter manifests itself therein that the human insulin molecule differs from other animal insulin molecules in a few differences in the composition of amino acid components.
Great improvements have been obtained as regards the insulin preparations after the introduction of the newest purification methods, but the formation of antibodies in the human body can still occur. It is believed that this can be remedied by using human insulin instead of other animal insulin.
It is known to prepare human insulin chemically, vide U.S. Pat. No. 3,903,068 and Hoppe-Seyler's Z. Physiol. Chem. 357, 759-767 (1976).
These processes comprise condensing a desoctapeptide-(B23-30) porcine insulin with a synthetic octapeptide corresponding to the positions B23-30 in human insulin. However, in the first process an alkaline hydrolysis is carried out, which is accompanied by unfavourable side reactions. The second process comprises a non-specific reaction giving rise to many side reactions and demanding complicated purification procedures. Consequently, these processes are not suitable for use on an industrial scale.
Moreover, U.S. Pat. No. 3,276,961 discloses a process for the preparation of human insulin from other animal insulins by the action of an enzyme, e.g. carboxypeptidase A or trypsin, in the presence of threonine. However, it has not proved to be possible to prepare human insulin to any appreciable extent by this known process. This is probably due to the fact that trypsin and carboxypeptidase A hydrolyze not only the lysyl-alanine peptide bond (B29--B30), but also other positions in insulin under the working conditions. Trypsin preferably hydrolyzes the arginyl-glycine peptide bond (B22--B23) rather than the lysyl-alanine bond (B29--B30). However, carboxypeptidase A cannot exclusively split off the alanine at the C-terminal of the B-chain without also splitting off asparagine at the C-terminal of the A-chain. It has later been shown that a specific condition, i.e. reaction in an ammonium bicarbonate buffer solution, is necessary in order to hinder the asparagine release, cfr. Hoppe-Seyler's Z. Physiol. Chem., 359, 799-802 (1978). Moreover, a considerable peptide formation scarcely occurs, since the velocity of the hydrolysis reaction is higher than that of the peptide synthesis at the working conditions.
It has later become known that addition of an organic solvent to the reaction medium in an enzymatically catalyzed process remarkably increases the velocity of the peptide bond synthesis and decreases the velocity of the hydrolysis, cfr. Ingalls et al. (1975), Biotechn. Bioeng. 17, 1627, and Homandberg et al., Biochemistry 17, 5220 (1978). The concentration of the solvent in the reaction medium should be high, and in the latter literature passage it is stated that when using 1,4-butanediol as solvent the best results are obtained with a concentration of 80% of said solvent.
Realizing this, desoctapeptide-(B23-B30) insulin (DOI) was successfully coupled by trypsin catalysis with a synthetic octapeptide corresponding to the B23-B30 positions of human insulin using an excess (10:1) of the latter reactant and using an organic solvent (DMF) in a concentration of more than 50%, cfr. J. Am. Chem. Soc. 101, 751-752 (1979). The coupling proceeds with a reasonable yield, but, all things considered, this process is still expensive and cumbersome, because it requires a trypsin catalyzed digestion of porcine insulin to form DOI and, moreover, the required octapeptide must be prepared by a complicated synthesis.
Moreover, Nature 280, 412-413 (1979) and Biochem. Biophys. Res. Com. 92 No. 2, 396-402 (1980) disclose a process for the semisynthetic preparation of human insulin, by which ala-B30 in porcine insulin is exchanged by threonine using trypsin or achromobacterprotease as catalyst. In this process porcine insulin is first hydrolyzed with carboxypeptidase or achromobacterprotease in the presence of NH.sub.4 HCO.sub.3 to form desalanine-B30 insulin (DAI). The trypsin or achromobacterprotease catalyzed coupling of DAI is carried out using a large excess of a protected threonine derivative, viz. threonine butyl ester (Thr-OBu.sup.t) in the ratio of 50:1 to 100:1 and in a high concentration of organic solvent, about 60% of a mixture of dimethylformamide and ethanol. Under such conditions the splitting of the Arg(B22)--Gly(B23) bond is greatly reduced.
It is apparent from the above disclosures that the disadvantages of the process known from U.S. Pat. No. 3,276,961 have been remedied by the reaction being carried out in high concentrations of organic solvents, and it has turned out that the increase in yield is relative to the large content of organic solvent. However, it is still an important feature that one of the reactants is present in a large excess. However, many of the proposed most suitable solvents are suspected of being mutagenic, and as they might be difficult to remove completely from the insulin product, the use of said solvents should be avoided as far as possible.